Method for identifying JNK and MLK inhibitors for treatment of neurological conditions

ABSTRACT

The present invention describes methods for identifying compounds that inhibit JNK and MLK kinase activity as drugs for treating a mammal susceptible to or having a neurological condition. This invention also discloses methods for preventing neuronal cell death and treating neurological conditions that involve neuronal cell death, particularly neurodegenerative diseases characterized by glutamine or kainate mediated toxicity, such as Huntington&#39;s disease and Alzheimer&#39;s disease.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 60/085,439, filed May 14, 1998, the entire teachings of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Excitotoxicity is related to excessive activation of glutamate receptorswhich results in neuronal cell death. The physiological function ofglutamate receptors is the mediation of ligand-gated cation channelswith the concomitant influx of calcium, sodium and potassium throughthis receptor-gated channel. The influx of these cations is essentialfor maintaining membrane potentials and the plasticity of neurons whichin itself plays a pivotal role in cognitive function of the centralnervous system. Li, H. B., et al., Behav. Brain Res., 83:225-228 (1997);Roesler, R., et al., Neurology, 50:1195 (1998); Wheal, H. V., et al.,Prog. Neurobiol., 55:611-640 (1998); Wangen, K., et al., Brain Res.,99:126-130 (1997). Excitotoxicity plays an important role in neuronalcell death following acute insults such as hypoxia, ischemia, stroke andtrauma, and it also plays a significant role in neuronal loss in AIDSdementia, epilepsy, focal ischemia. Coyle, J. T. & Puttfarken, P.,Science, 262:689-695 (1993). Neurodegenerative disorders, such asHuntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease(PD) and amyotrophic lateral sclerosis (ALS), are characterized by theprogressive loss of a specific population of neurons in the centralnervous system. Growing evidence suggests that glutamate-mediatedexcitotoxicity may be a common pathway which contributes to neuronalcell death in a wide range of neurological disorders. Coyle, J. T. &Puttfarken, P., Science, 262:689-695 (1993).

The molecular mechanisms of excitotoxicity-mediated neuronal cell deathremains obscure. Over-production of free radicals that lead toimpairment of mitochondrial function is the most widely held hypothesis.Beal, M. F., et al., Ann. Neurol., 38:357-366 (1995); Coyle, J. T. &Puttfarken, P., Science, 262:689-695 (1993). However, it is unclearwhether the increase of free radicals is the precursor that initiatesneuronal degeneration or, rather, a subsequent consequence of neuronaldegeneration. Interestingly, administration of antioxidants has littleneuroprotective effect in patients suffering from variousneurodegenerative diseases. Shults, C. W., et al., Neurology, 50:793-795(1998). Thus, some other mechanism(s) must exist forexcitotoxicity-induced neuronal cell death.

c-Jun N-terminal kinases (JNKs) are identified as kinases which areactivated upon stimulation by various environmental stimuli such as UVlight, γ irradiation and mitogenic signals. Hibi, M., et al., GenesDev., 7:2135-2148 (1993); Kyriakis, J. M., et al., Nature, 369:156-160(1994). The precise biological function of JNKs remains to be explored.However, some recent reports suggest that JNKs are involved in neuronalapoptosis induced by deprivation of survival factors, i.e., neurotrophicfactors which support neuronal survival. Ham, J., et al., Neuron,14:927-939 (1995).

Mixed-lineage kinases (MLKs), so called because these proteins containstructural domains associated with a variety of cell types, were clonedfrom a cDNA library derived from mRNA from cancer tissue, MLKs wereinitially thought to participate in the oncogenesis of some cancers,although high levels of expression of MLKs were found in the normalbrain. Dorow, D. S., et al., Eur. J. Biochem., 213:701-710 (1993);Dorow, D. S., et al., Eur. J. Biochem., 234:492-500 (1995).

Searching for biochemical targets which are amenable to screening forneuroprotective therapeutic agents is of central concern in neurosciencetoday. However, no clinically available pharmaceutical tool to date isemployed for blocking excitotoxicity and preventing neuronal cell lossin various neurological disorders due to a lack of suitable biochemicaltargets. Glutamate receptor antagonists, such as MK-801, althoughsuccessful in protecting neurons in animal experiments, have all failedin the clinical setting due to their blockage of cognitive functionmediated by the receptors, as well as high toxicity to the centralnervous system. Thus, an understanding of the molecular mechanism(s) ofneuronal cell death induced by excitotoxicity is essential for theidentification of new biochemical targets and the establishment ofreliable methods for screening new therapeutic drugs from chemicallibraries that can be utilized in the treatment of a variety ofneurological disorders.

SUMMARY OF THE INVENTION

This invention relates to the discovery that inhibiting a JNK or MLKwithin a hippocampal neuronal cell can protect the cell from apoptosis.As such, JNK and MLK can be used as drug targets to screen fortherapeutic agents to prevent glutamate or kainic acid mediatedtoxicity, to block excitotoxicity and to prevent neuronal loss in avariety of neurological conditions, such as Huntington's disease andAlzheimer's disease.

In one aspect of the invention, a method is described for assessing acompound's ability to inhibit neuronal cell death, and thus to identifycompounds that can be used to prevent and/or treat neurologicalconditions. According to the method, neuronal cells having activated MLKand/or JNK activity are contacted with a compound and the number ofneuronal cells that die is determined. A decrease in the number of deadneuronal cells in the presence of the compound compared to the number ofdead neuronal cells in the absence of the compound is indicative of thecompound's ability to inhibit neuronal cell death. Preferably, theneuronal cells are apoptotic neurons (i.e., cell death caused by aneurological condition) or neurons that are induced to undergoapoptosis, such as by contacting the neuronal cells with neurotoxin(e.g., glutamate, quinolinic acid or kainic acid); or by geneticmanipulation of the neuronal cells. Most preferred are HN33 hippocampalneuronal cells.

In another embodiment, the invention features a method for testing acompound's potential as a drug for treating a mammal (e.g., a human)susceptible to or having a neurological condition by (1) contacting acompound with a JNK (e.g., JNK3) or MLK (e.g., MLK2); (2) measuring thelevel of a JNK-associated or MLK-associated activity (e.g., a kinaseactivity); and (3) comparing the level of the JNK-associated orMLK-associated activity in the presence of the compound with the levelof the JNK-associated or MLK-associated activity in the absence of thecompound. The compound is a potentially useful drug for treating themammal when the level of the JNK-associated or MLK-associated activityin the presence of the compound is less than the level of theJNK-associated or MLK-associated activity in the absence of thecompound.

The JNK or MLK can be within a cell, which can be an animal (e.g.,human) cell in vivo. When the JNK or MLK is within a cell, theJNK-associated or MLK-associated activity can be apoptosis, which can bemeasured by a TUNEL assay (described below). Apoptosis within such acell can be induced by introducing into the cell a huntingtin proteinthat has at least 40 consecutive glutamic acids (e.g., polyglutaminestretch-expanded huntingtin). Alternatively, apoptosis can be induced byintroducing into the cell the C-terminal 100 amino acids of an amyloidprecursor protein (APP). Preferably, the huntingtin protein or theamyloid precursor protein is introduced by a vector, especially anucleic acid vector. When the cell is within an animal, theJNK-associated or MLK-associated activity can be neurodegeneration.

The invention also features a method for testing a compound's potentialas a drug for treating a mammal (e.g., a human) susceptible to or havinga neurological condition by (1) contacting a compound with a neuronalcell containing a JNK (e.g., JNK3) or MLK (e.g., MLK2); (2) measuringthe level of a JNK or MLK protein activity (e.g., kinase activity, suchas the presence or amount of phosphorylated product) in the cell; and(3) comparing the level of the JNK or MLK protein activity in the cellin the presence of the compound with the level of the JNK or MLK proteinactivity in the cell in the absence of the compound. The compound is apotentially useful drug for treating the mammal when the level of theJNK or MLK protein activity in the cell in the presence of the compoundis less than the level of the JNK or MLK protein activity in the cell inthe absence of the compound. Alternatively, cell viability can beascertained by determining the degree of neuronal cell death, wherein adecreased number of dead neuronal cells in the presence of the compoundcompared to the number of dead neuronal cells in the absence of thecompound is indicative of the compound's ability to inhibit JNK or MLKprotein activity, thereby preventing neuronal cell death.

In another aspect, the invention provides a method for testing thepotential of a JNK or MLK inhibitor as a drug for treating a mammal(e.g., a human) susceptible to or having a neurological condition. Themethod can be performed on compounds identified as JNK and/or MLKinhibitory agents using the methods of this invention to confirm theirinhibitory effectiveness under apoptotic conditions. Accordingly, themethod provides (1) incubating a neuronal cell in the presence of a JNKor MLK inhibitor; (2) contacting surviving cells with an agent thatinduces apoptosis in the cell; and (3) comparing the occurrence ofapoptosis in the cell in the presence of the JNK or MLK inhibitor withthe occurrence of apoptosis in the cell in the absence of the JNK or MLKinhibitor. The compound is a potentially useful drug for treating themammal when the occurrence of apoptosis in the cell in the presence ofthe JNK or MLK inhibitor is less than the occurrence of apoptosis in thecell in the absence of the JNK or MLK inhibitor.

The methods of the invention are used to identify inhibitors of JNK orMLK which are potentially useful for the treatment of a neurologicalcondition, including neuronal cell death following acute insults such ashypoxia, ischemia, stroke, and trauma. Other neurological conditionstreatable with compounds identified by the methods of the inventioninclude AIDS dementia, epilepsy, focal ischemia, Huntington's disease,Alzheimer's disease, Parkinson's disease, and amyotrophic lateralsclerosis. Each of these conditions are characterized by the progressiveloss of a specific population of neurons in the central nervous system.The methods of the invention are particularly useful in findingcompounds which can be used to prevent and/or treat neurologicalconditions, including genetic neurological conditions. The inventionalso pertains to compounds, identified using the methods describedherein, that inhibit MLK and/or JNK activity and that prevent neuronalcell death occurring in a mammal susceptible to or having a neurologicalcondition, particularly neurodegenerative diseases, such as Huntington'sdisease and Alzheimer's disease.

The invention also provides methods for preventing and/or treatingneuronal conditions in a mammal comprising administering to a mammal, inneed thereof, an effective therapeutic amount of a compound thatinhibits JNK and/or MLK. The inhibitory effects of the compound willreduce and/or prevent neuron cell death occurring in a mammalsusceptible to or having a neurological condition. In a preferredembodiment, the neurological condition is a neurological disease wherebyglutamate or kainic acid mediated excitotoxicity is involved in neuronalcell death. JNK and/or MLK inhibitors identified using any of themethods described herein are useful as therapeutic or prophylatic drugsto prevent neuronal loss.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating a time course of cell death uponexpression of normal or mutated huntingtin in HN33 cells. pcDNA1 (solidbox), pFL16HD (circles), pFL48HD (triangles) and pFL89HG (open boxes).

FIG. 2 is a graph illustrating apoptotic cell death induced byexpression of mutated huntingtin with 48 or 89 polyglutamine repeats wascompletely blocked by added the ICE or CPP32 inhibitor in the mediumindicating HN33 cells are undergoing apoptotic cell death.

FIG. 3 is a graph illustrating the fold of increased JNK activity inHN33 cells upon expression of mutated huntingtin with 48 or 89polyglutamine repeats.

FIG. 4 is a graph illustrating apoptotic cell death of HN33 cellsinduced by mutated huntingtin with either 48 or 89 polyglutamine repeatswas inhibited by co-expression of dominant negative mutant form of SEK1but not wild-type SEK1.

FIG. 5 is a graph illustrating apoptotic cell death of HN33 cellsinduced by the treatment of glutamate (250 μM) or kainic acid (kainate,200 μM) was significantly attenuated by expression of dominant negativemutant form of SEK1 (K54R) but not wild-type SEK1, indicating thatglutamate or kainate induced the activation of the SEK1-JNK pathway tomediate neuronal apoptosis.

FIG. 6 is a graph illustrating the fold of increased JNK activity inHN33 cells upon stimulation of glutamate or kainate receptor indicatingthat glutamate or kainate receptor activation also stimulated the JNKactivity like expression of mutated huntingtin.

FIG. 7 is a graph illustrating a time course of cell death uponfollowing transfection of pcDNA1 (control) APP or APP deletion mutantAPP-C-100, pcDNA1 (open box), wild-type APP (solid diamond), APP-C-100(solid circles).

FIG. 8 is a graph illustrating the fold of increased JNK activity inHN33 cells following transfection of pcDNA1 (control) APP or APPdeletion mutant APP-C-100, indicating that expression of APP-C-100stimulated the JNK activity.

FIG. 9 is a graph illustrating apoptotic cell death of HN33 cellsinduced by expression of APP-C-100 was significantly attenuated byco-expression of dominant negative mutant form of SEK1 (K54R) but notwild-type SEK1, indicating that amyloid precursor protein induced theactivation of the SEK1-JNK pathway to mediate neuronal apoptosis.

FIG. 10 is a graph illustrating a time course of cell death followingtransfection of pRK5CMV (control), wild-type MLK2 or kinase dead versionof MLK2, pRK5CMV (open box), wild-type MLK2 (solid diamond), kinase deadMLK2 (solid circles).

FIG. 11 is a graph illustrating apoptotic cell death of HN33 cellsinduced by expression of MLK2 was significantly attenuated byco-expression of dominant negative mutant form of SEK1 (K54R) but notwild-type SEK1, indicating that MLK2 induced the activation of theSEK1-JNK pathway to mediate neuronal apoptosis.

FIG. 12 is a graph illustrating apoptotic cell death of HN33 cellsinduced by expression mutated huntingtin with 48 or 89 CAG repeats wasblocked by co-expression of kinase dead MLK2, indicating that theMLK2-associated activity mediated neuronal cell death in Huntington'sdiseases.

FIG. 13 is a graph illustrating apoptotic cell death of HN33 cellsinduced by the treatment of glutamate (250 μM) or kainic acid (kainate200 μM) was blocked by expression of kinase dead MLK2 indicating thatthe MLK2-associated activity mediated neuronal cell death in neuronalexcitotoxicity induced by glutamate or kainate receptor activation.

FIG. 14 is a graph illustrating apoptotic cell death of HN33 cellsinduced by expression of deletion APP mutant APP-C-100 was blocked byco-expression of kinase dead MLK2, indicating that the MLK2-associatedactivity mediated neuronal cell death in Alzheimer's diseases.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the discovery that two families of proteins,JNK and MLK, can serve as targets for the treatment of neurologicalconditions. It has been discovered that inhibition of JNK3, a member ofthe JNK family, and MLK2, a member of the MLK family, can protect aneuronal cell from apoptosis induced by polyglutamine-expandedhuntingtin, whose expression caused HD in humans. The Huntington'sDisease Collaborative Research Group, Cell, 72:971-983 (1993). Thus, theinvention provides a method for assessing compounds for their potentialas drugs for the treatment of neurological conditions, such asHuntington's disease, by determining whether the compound can inhibit aJNK- or MLK-associated activity.

MLK and JNK participate in a biochemical cascade (activation ofMLK-SEK1-JNK1) that mediates neuronal cell toxicity. Upon stimulation byglutamate, kainic acid, or other receptor agonists, the glutamatereceptor, located at the cell surface, is activated and interacts withassociated proteins (e.g., PDZ domain-containing proteins) whose SH3domain in turn binds to a MLK protein, thereby activating its kinaseactivity. The MLK protein directly binds to and stimulates a SEK1protein which in turn binds to and stimulates JNK. Over stimulation ofJNK can lead to neuronal apoptosis (cell death). Normally MLK isinhibited by a protein, such as normal huntingtin, binding to its SH3domain thus inhibiting the enzyme's kinase activity. This inhibitionprevents the formation of the cascade resulting in no or little JNKactivity, thereby preserving neuronal cell viability.

To determine whether polyglutamine-expanded huntingtin induced celltoxicity in neurons, the expression of huntingtins with 16, 48 or 89 CAGrepeats in an immortalized rat hippocampal neuronal cell line (HN33) wasexamined. HN33 has been described in detail in Hammond, D. N., et al.,Brain Res., 512:190-200 (1990); the entire teachings of which areincorporated herein by reference. The hippocampal neurons serve as apotential target of mutated huntingtin, mutated amyloid precursorprotein (APP), as well as glutamate stimulation. The resultsdemonstrated that expression of mutated huntingtin with 48 or 89 CAGrepeats stimulated JNKs and induced apoptotic cell death in HN33 cells,while expression of normal huntingtin with 16 CAG repeats had no toxiceffect. JNK activation occurs several hours prior to neuronal apoptosissuggesting that it is an early signal for the induction of neuronalapoptosis. Furthermore, co-expression of a dominant negative form ofstress signaling kinase (SEK1), which serve as a specific inhibitor ofJNKs, attenuated both JNK activation and neuronal apoptosis induced bymutated huntingtin. This study demonstrates that JNK activation canmediate neuronal cell death in neurological disorders. Like mutatedhuntingtin, the treatment of HN33 cells with glutamate also resulted inJNK activation and apoptotic cell death. Expression of dominant negativemutant SEK1 significantly attenuated glutamate-induced toxicity. Takentogether, these studies indicate that JNK is a common cellular mediatorfor induction of neuronal cell death mediated by both glutamate andmutated huntingtin.

The mechanism for how mutated huntingtin stimulates JNKs was alsoexamined. The huntingtin protein contains multiple SH3 domain bindingsites. In previous studies, it has been demonstrated that normalhuntingtin directly binds to SH3 domain-containing proteins Grb2 andRasGAP. Liu, Y. F., et al., J. Biol. Chem., 272:8121-8124 (1997). MLK1,MLK2 and MLK3 (MLKs) are the only known kinases that directly activatethe SEK1-JNK cascade and contain a SH3 domain as well as a SH3 domainbinding site. MLK2 is a neuronal form of MLKs. The presence of MLKs, inparticular MLK2, appears to be required for mutated huntingtin-mediatedJNK activation and neuronal apoptosis. MLKs were expressed in HN33 cellswhere expression of mutated huntingtin induced JNK activation andapoptosis, while in both 293 and CHO cells where MLKs are absent, theexpression of mutated huntingtin did not generate any cell toxicity in293 and CHO cells. However, co-expression of mutated huntingtin alongwith MLK2 in 293 cells caused rapid apoptotic cell death. The SH3 domainof MLKs is required for their proper cellular localization andactivation of the SEK1-JNK pathway. In the in vitro binding-studies, itwas shown that normal huntingtin binds to the SH3 domain of MLK2 andinhibits enzyme activity while expansion of the polyglutamine repeatsinterferes with huntingtin's binding and consequently is not inhibitorywith respect to MLK2's activity. Furthermore, expression of kinase deadMLK2 completely blocks mutated huntingtin-induced neuronal cell death.These studies indicate that mutation of huntingtin results in anincrease of free MLKs thereby causing over-activation of theMLK-SEK1-JNK cascade which ultimately leads to neuronal cell death.

The SH3 binding motif is found in six other proteins involved inpolyglutamine repeat-expanded neurodegenerative hereditary diseases,such as ataxia-1, ataxia-2, ataxia-6, ataxia-7, Kennedy disease,dentatorubral and pallidoluysian atrophy (DRPLA). The normal (wild-type)counter-part proteins bind to and suppress MLK activity, in contrast tothe mutated protein form which lose such ability, resulting in theover-activation of the MLK2-SEK1-JNK pathway in neurons.

The JNK kinases phosphorylate and activate the transcription factorc-Jun which mediates apoptosis. Recent studies suggest that c-Jun mayserve as an important mediator for neuronal apoptosis induced by avariety of environmental stresses. In primary cultured sympathetic,hippocampal or cerebellar granule neurons, deprivation of growth factorin these primary cultures lead to persistent activation of JNKs andconsequently the phosphorylation of c-Jun. Ham, J., et al., Neuron,14:927-939 (1995); Xia, Z., et al., Science, 270:1326-1330 (1995).Suppression of c-Jun expression by antisense-oligonucleotides, orfunctional blockade by microinjection of antibodies or expression ofdominant negative c-Jun prevents neuronal apoptosis; in contrast,over-expression of c-Jun induces apoptosis in sympathetic neurons.Estus, S., et al., J. Cell Biol., 127:1717-1727 (1994); Ham, J., et al.,Neuron, 14:927-939 (1995). Enhanced c-Jun expression occurs indegenerating and apoptotic neurons after ischemia, nerve fibertransection, axotomized brains, UV irradiation and various other typesof neuronal injury. Ferrer, I., et al., Eur. J. Neurosci., 8:1286-1298(1998); Herdegen, T., et al., J. Neurosci., 18:5124-5135 (1998).

Increased c-Jun expression and activation are also implicated in thegeneration of the neuronal apoptotic process induced by glutamate orkainic acid. Administration of glutamate or quinolinic acid, aN-methyl-D-aspartate (NMDA) receptor agonist, or kainic acid in ratsresults in a rapid induction of c-Jun expression and neuronal apoptosis.Coyle, J. T. & Puttfarken, P., Science, 262:689-695 (1993); Qin, Z.-H.,et al., Mol. Pharmacol., 53:33-42 (1997). These physiological events canbe blocked by the NMDA receptor antagonist MK-801. Qin, Z.-H., et al.,Mol. Pharmacol., 53:33-42 (1997).

Over-activation of the c-Jun-mediated JNK cascade has also beenimplicated in various other neurodegenerative disorders. Enhanced c-Junexpression is observed in the brains from patients suffering frommultiple sclerosis, AD and ALS. Anderson, A. J., et al., Exp. Neurol.,125:286-295 (1994); Martin, D. G., et al., Neurosci. Lett., 212:95-98(1996). These studies support the basis of the present invention in thatthe over-activation (or stimulation) of the MLK-SEK1-JNK cascade leadsto the increase of expression, activation and translocation of c-Junwhich is responsible for neuronal cell death in these, and other,neurodegenerative disorders. Thus, inhibition of this cascade canprotect neurons from toxicity induced by endo- and/or exo-toxinsincluding, but not limited to, mutated proteins like huntingtin,quinolinic acid, kainic acid, glutamate over-excitation as well as otheretiological agents.

Based upon these findings, JNK and MLK can be used as targets for thedevelopment of inhibitory compounds of JNK- and MLK-associated activity,and such compounds can be used to prevent neuronal loss, such as inducedby excitotoxicity or glutamate- or kainic acid-mediated toxicity. Asused herein, a “JNK-associated activity” is any biochemical, cellular,or physiological property that varies with any variation in JNK genetranscription or translation, or JNK protein activity. Likewise, a“MLK-associated activity” is any biochemical, cellular, or physiologicalproperty that varies with any variation in MLK gene transcription ortranslation, or MLK protein activity. A JNK or MLK inhibitor is acompound that inhibits a JNK or MLK protein activity. A JNK or MLKprotein activity is any measurable biochemical activity possessed by theprotein, e.g., a kinase activity or an ability to bind to anotherprotein such as c-Jun.

Inhibitors of MLKs identified by the methods described herein can blockpersistent activation of glutamate receptor-induced over-activation ofMLKs without affecting other receptor functions, such as the involvementof neuronal plasticity and cognitive functions. Inhibition of MLKs willattenuate the JNK activity in neurons and protect neurons fromexcitotoxicity thereby preventing neuronal loss in these diseases.Inhibitors of JNKs or MLKs identified by the methods described hereincan suppress glutamate receptor-induced activation of the MLK-SEK1-JNKcascade and prevent neuronal apoptosis in various neurological diseases.

The term “neurological condition” as used herein is intended to embracedisorders, disease states and disturbances which cause or result inneuronal cell injury, compromise or cell death. Neurological conditionscan result from axonal degeneration, ischemia due to stroke, heartarrest, exposure, exposure to neurotoxins such as, but not limited to,glutamate, kainic acid and quinolinic acid, MPTP exposure to bacterialor viral toxins, impaired function or dysfunction of neurons such asincrease or decrease of neurotransmitter synthesis and/or release.Neurological diseases and disturbances include, but are not limited to,Alzheimer's disease; Parkinson's disease; motor neuron diseases such asamyotrophic lateral sclerosis (ALS), Huntington's disease andsyringomyelia; ataxias, dementias; chorea; dystonia; dyslinesia;encephalomyelopathy; parenchymatous cerebellar degeneration; Kennedydisease; Down syndrome; progressive supernuclear palsy; DRPLA, stroke orother ischemic injuries; thoracic outlet syndrome, trauma; electricalbrain injuries; decompression brain injuries; AIDS dementia; multiplesclerosis; epilepsy; concussive or penetrating injuries of the brain orspinal cord; peripheral neuropathy; brain injuries due to exposure ofmilitary hazards such as blast over-pressure, ionizing radiation, andgenetic neurological conditions. By “genetic neurological condition” ismeant a neurological condition, or a predisposition to it, that iscaused at least in part by or correlated with a specific gene ormutation within that gene; for example, a genetic neurological conditioncan be caused by or correlated with more than one specific gene.Examples of genetic neurological conditions include, but are not limitedto, Alzheimer's disease, Huntington's disease, spinal and bulbarmuscular atrophy, fragile X syndrome, FRAXE mental retardation, myotonicdystrophy, spinocerebellar ataxia type 1, dentatorubral-pallidoluysianatrophy, and Machado-Joseph disease.

In one aspect of the invention, a method is described for assessing acompound's ability to inhibit neuronal cell death. According to themethod, neuronal cells having activated MLK and/or JNK activity arecontacted with a compound and the number of neuronal cells that die isdetermined. A decrease in the number of dead neuronal cells in thepresence of the compound compared to the number of dead neuronal cellsin the absence of the compound is indicative of the compound's abilityto inhibit neuronal cell death. Preferably, the neuronal cells areapoptotic neurons or neurons that are induced to undergo apoptosisneuronal cells with neurotoxin or genetic manipulation.

A neuronal cell useful in the methods of the invention is preferablysusceptible to JNK-dependent or MLK-dependent apoptosis. To facilitateapoptosis, such a cell can express a polypeptide known to be associatedwith or induce a neurodegenerative disease, such as apolyglutamine-expanded polypeptide (e.g., polyglutamine-expandedhuntingtin) or the C-terminal 100 amino acid fragment of an amyloidprecursor protein. A preferred neuronal cell that is useful forassessing MLK and/or JNK inhibitors is an immortalized rat hippocampalneuronal cell line HN33, with or without genetic manipulations to induceapoptosis, as described above.

In another embodiment, the invention features a method for testing acompound's potential as a drug for treating a mammal (e.g., a human)susceptible to or having a neurological condition by (1) contacting acompound with a JNK (e.g., JNK3) or MLK (e.g., MLK2); (2) measuring thelevel of a JNK-associated or MLK-associated activity (e.g., a kinaseactivity); and (3) comparing the level of the JNK-associated orMLK-associated activity in the presence of the compound with the levelof the JNK-associated or MLK-associated activity in the absence of thecompound. The compound is a potentially useful drug for treating themammal when the level of the JNK-associated or MLK-associated activityin the presence of the compound is less than the level of theJNK-associated or MLK-associated activity in the absence of thecompound.

In another aspect of the invention, a putative inhibitory agent isincubated in vitro in the presence of JNK and appropriate JNKsubstrates, such as c-Jun and a phosphate donor like adenosinetriphosphate (ATP), under conditions sufficient for enzymatic activity;followed by isolating the phosphorylated product. Isolated JNK protein,including JNK1, JNK2 and JNK3, can be obtained for this, as well asother assays, by several different molecular and chromatographic methodsknown to those skilled in the art. The JNK polypeptides useful in themethods of the present invention are preferably wild-type whose sequenceis known and readily available. The human JNK3 polypeptide is describedby Martin et al., Mol. Brain Res., 35:47-57 (1996). Other JNK proteinsuseful in the methods of the invention include those described inGenBank Accession Nos. U17743, U49249 and AF006689. Isolated JNKprotein, from about 0.5 μg to about 2 μg of purified JNK, is incubatedwith substrate in an aqueous medium, such as a kinase buffer (containingabout: 20 mM HEPES, pH 7.5, 15 mM MgCl₂, 15 mM β-glycerophosphate, 0.1mM Na₂PO₄ and 2 mM dithiothreitol) at about 30° C. for approximately 15minutes. The substrates that can be used in this reaction include, butare not limited to, c-Jun, from about 1 μg to about 3 μg, a knownsubstrate for JNK's kinase activity, and the phosphate donor, ATP(approximately 2.5 mM). For detection purposes, 5 μCi of [γ-³²]ATP canbe used as a co-substrate. The assay system can also include in theincubation mixture a putative inhibitory JNK agent. The reaction can beterminated by addition of Laemmeli buffer, approximately 20 μL. Theaddition of this buffer will also prepare the sample for productanalysis. The reaction mixture can be subjected to sodium dodecylsulfatepolyacrylamide gel electrophoresis (hereinafter SDS-PAGE) in order todetermine the amount of phosphorylated c-Jun that was formed in thereaction. The radioactivity emitted from the γ³²P can be measured usingconventional radioactivity gel detection systems, such as an X-rayfollowed by β-scan. The phosphorylated c-Jun product will have adifferent migration rate along the gel when compared to the labeled ATPco-substrate and therefore will not be confused with the kinase product.A determination can then be made concerning whether the test agentinhibited JNK's activity by comparing reaction mixtures having the agentpresent to reaction mixtures without addition of the compound.

Alternatively, JNK substrates, such as c-Jun and ATP, can be incubatedin the presence of a cellular extract containing putative JNK enzymeactivity, including JNK1, JNK2 and JNK3. An inhibitory agent to betested can be placed in the reaction vial along with the other reactantsto examine the efficacy of the agent. The reaction and detectionprotocol can be conducted in the same manner as that describe above forthe in vitro assay without cellular extract. The cellular extract canoriginate from a cell or tissue culture system, or can be prepared fromwhole tissue employing isolation and purification protocols known tothose skilled in the art.

In another embodiment, the invention pertains to contacting a cell witha putative inhibitory agent in order to screen for inhibitory agents ofJNK activity, including JNK1, JNK2 and JNK3. The cell to be contactedcan be of a cell or tissue culture system. The putative inhibitory agentis delivered to the cell under conditions sufficient for enzymaticactivity in any of a number of ways known to those skilled in the art.If the agent is not membrane permeable, then the agent can be deliveredinto the cell via electroporation, or if it is a polypeptide, a nucleicacid or viral vector can be employed. If the cell has JNK activitypresent in an active form, then JNK can be stimulated by delivering tothe cell SEK1, a known stimulator of JNK. If the cell lacks a JNK geneor functional JNK gene or transcript or translational product, the cellcan be transfected with an operatively linked JNK gene. “Operativelylinked” is intended to mean that the nucleotide sequence is linked to aregulatory sequence in a manner which allows expression of the nucleicacid sequence.

To detect the phosphorylated product, any number of protocols known tothose skilled in the art can be used including, but not limited to,Western blot analysis and apoptosis analysis. Antibodies, bothmonoclonal and polyclonal, can be made against epitopes derived from thesite on the JNK substrate bound to a phosphate group. A SDS-PAGEprocedure can be performed on homogenized cell extract and subsequentlysubjected to Western blot analysis using an antibody specific for aphosphorylated JNK substrate, such as c-Jun.

An apoptosis analysis can also be performed in order to determine whateffect, if any, the putative inhibitory agent has on JNK-associatedactivity. For example, an expression vector encoding JNK3 is transfectedinto an appropriate target cell to induce apoptosis. Target cells arecells that are susceptible to apoptosis. Rat hippocampal neuronal cellline HN33 is a preferred target cell. Alternatively, target cells whichnaturally contain JNK can be used. In either event, the target cells arecultured in the presence or absence of a test agent and the occurrenceof apoptosis determined using known techniques. For example, stainingthe cell with Hoechst 3342 (Sigma Chemical Co.) and observing thestained cell under the microscope. Apoptotic cells appear containingclearly segmented, condensed chromatin. Alternatively, apoptosis can bedetermined by using the TUNEL assay as described by Thomas, L. B., etal., Exp. Neurol., 133:265-272 (1995). See also U.S. Pat. No. 5,593,879,for techniques for examples of stains used to distinguish apoptoticcells.

In another embodiment, the invention pertains to a method for screeningpotential inhibitory agents of JNK activity, including JNK1, JNK2 andJNK3, by administering to an animal, including mammals, the agent anddetermining what effect, if any, the agent has on the animal'sphysiological status. The animal is given an amount of test agentsufficient to allow for proper pharmacodynamic absorption and tissuedistribution in the animal. Preferably, the animal used is an example ofa model system mimicking a neurological condition. However, to test thesafety of the putative agent, a normal animal is preferably alsosubjected to the treatment. Following administration of the agent, theanimal can be sacrificed and tissue sections from the brain, as well asother tissues, can be harvested and examined for apoptosis using, forexample, the TUNEL assay. Yang, D. D., et al., Nature, 389:865-870(1997). In another embodiment, an animal model afflicted with aneurological condition (e.g., neurodegenerative disorder) can beadministered a JNK and/or MLK inhibitor and the symptoms associated withthe neurological condition are evaluated. Attenuation, amelioration orimprovement of the neurodegenerative symptoms can be assessed, wherebyimprovement is indicative of the inhibitors ability to prevent and/ortreat the neurological condition.

The methods described above can be likewise employed to identify/screenfor inhibitory agents of MLK-associated activity, including MLK1, MLK2and MLK3. Appropriate MLK substrates include, but are not limited to,ATP and SEK1, a protein known to activate JNKs by phosphorylation. TheMLK polypeptides useful in the methods of the present invention arepreferably wild-type whose sequence is known and readily available. Thehuman MLK2 polypeptide is described by Dorow, D. S., et al., Eur. J.Biochem., 234:491-500 (1995). Another MLK protein useful in the methodsof the invention is described in GenBank Accession No. L32976.

The JNK and MLK useful in the methods of the invention are not limitedto the naturally occurring sequences described above. JNK and MLKcontaining substitutions, deletions, or additions can also be used,provided that those polypeptides retain at least one activity associatedwith the naturally occurring polypeptide and are at least 70% identicalto the naturally occurring sequence.

To determine the percent identity of two polypeptide sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the sequence of a first amino acid sequence). The aminoacid residues at corresponding amino acid positions are then compared.When a position in the first sequence is occupied by the same amino acidresidue as the corresponding position in the second sequence, then themolecules are identical at that position. The percent identity betweenthe two sequences is a function of the number of identical positionsshared by the sequences (i.e., % identity=# of identical positions/total# of positions×100).

The determination of percent homology between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin et al., Proc. Natl. Acad. Sci. USA,90:5873-5877 (1993). Such an algorithm is incorporated into the NBLASTand XBLAST program, score=50, wordlength=3 to obtain amino acid sequencehomologous to protein molecules useful in the methods of the invention.To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., Nucleic Acids Res,25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov. Another preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers et al., CABIOS (1989).Such an algorithm is incorporated into the ALIGN program (version 2.0)which is part of the GCG sequence alignment software package. Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used.

The percent of identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

An example of a JNK or MLK that is not naturally occurring, thoughuseful in the methods of the invention, is aJNK-gluthathione-S-transferase (JNK-GST) fusion protein. Such a proteincan be produced in large quantities in bacteria and isolated. The JNKfusion protein can then be used in an in vitro kinase assay in thepresence or absence of a candidate drug for treating neurologicalconditions.

The present invention also pertains to methods for testing theeffectiveness of inhibitory compound identified using the methods ofthis invention for neuronal protection for the prevention and/ortreatment of a variety of neurological disorders. In one embodiment ofthe invention, the effectiveness of neuronal protection by JNK or MLKinhibitors against excitotoxicity stimuli can be assessed by thepre-treatment of HN33 cells with the identified JNK or MLK inhibitorsprior to stimulation with glutamate, or other neurotoxins such as kainicacid, MPP, quinolinic acid or transfection of the mutated form ofhuntingtin or amyloid precursor protein (APP). For example, HN33 cells,plated on a 96-well plate, are grown in DMEM-F12 medium and prior toexperimentation, the medium is removed and cells are washed once withserum-free medium. 0.5 ml of serum free medium is added and cells areincubated at 37° C. cell culture incubator for 10 minutes. In the caseof the treatment with glutamate or other neurotoxins to induceapoptosis, the identified JNK or MLK inhibitor is added to theserum-free medium and incubation continued for another 5 minutes. Thenglutamate or neurotoxin are added to the medium. If the JNK or MLKinhibitor is effective, the amount of apoptotic cells will besignificantly reduced or totally inhibited, as compared with theappropriate control. Such a result indicates that these inhibitors areeffective for the prevention of neuronal death in various neurologicaldisorders. In the case of expression of mutated huntingtin or APP-C-100(vector which expresses the c-terminal 100 amino acids of APP) to induceapoptosis, the JNK or MLK inhibitor is added during or 2-6 hours aftertransfection. In a particular embodiment, the IC₅₀ of the JNK or MLKinhibitors in suppression of neuronal apoptosis can be also assessed bythis 96-well based assay. In this case, different concentrations of JNKor MLK inhibitor are added to the medium prior to the treatment withglutamate or other neurotoxins or during transfection of the mutatedhuntingtin or APP to establish a pharmacological profile for eachinhibitor. The IC₅₀ of each inhibitor is a very important value fordesigning further study of the effectiveness in different animal modelsand for directing clinical trials of these inhibitors.

The present invention also pertains to methods for the prevention ortreatment of neurological conditions, either through prophylaticadministration prior to the occurrence of an event known to cause aneurological condition or therapeutic administration immediatelyfollowing the event and periodically thereafter. Such prophylatic andtherapeutic treatments are intended to prevent neuronal cell death orreduce the degree of cell death. Given the involvement of JNK and MLK inthe cascade leading to neuronal cell death, these two kinases presenttargets for a therapeutic regime. According to the method, a mammal,including human, is administered an effective therapeutic amount of anagent that targets JNK- and/or MLK-associated activity. A therapeuticamount for a given agent is that amount administered to achieve thedesired result, for example, the inhibition of kinase activity in eitherJNK or MLK or both, or attenuation, amelioration of or improvement inthe symptoms associated with the neurological condition.

In one embodiment, the JNK-associated activity that is targeted is JNK'skinase activity. By inhibiting JNK's activity with an agent, neuronalcell death can be avoided. The JNK activity to be targeted includesJNK1, JNK2 and JNK3. In another embodiment, the enzyme activity targetedis MLK. If MLK is not inhibited, then it will directly bind to andphosphorylate SEK1 resulting in its activation which in turn willstimulate JNK, thereby causing neuronal cell death. By inhibiting MLKactivity, including MLK1, MLK2 and MLK3, the SEK1 phosphorylation andconcomitant stimulation can be eliminated, thereby saving neuronal cellsfrom apoptosis. This therapeutic approach can be used to prevent and/ortreat neurological conditions, as described above. The inhibitory agentsidentified using the methods described herein are particularly usefulfor suppressing glutamate receptor-induced activation of JNK,glutamate-mediated toxicity and apoptosis caused by excitotoxicity.

Compounds identified using the methods described herein are designed toselectively inhibit the neuronal isoform of kinase which is involved inneuronal loss in neurodegenerative diseases. These kinase inhibitorswill selectively decrease a specific kinase activity in neurons andprotect neurons from a variety of oxidative stimuli thereby allowing abroad range of clinical applications. Because the neuronal isoform ofkinase is selectively attenuated, side effects in peripheral tissues maybe neglectable and because other isoforms of the kinase are present inneurons and will provide complementary function for the inhibitedisoforms of the kinase, side effects in the central nervous system mayalso be minimal. A specific inhibitor of MLK2 or JNK3 should be aneffective, low toxic neuroprotective drug for the treatment of a widerange of neurodegenerative disorders. In particular, two differentkinases on the same signaling pathway can be targeted. These differentkinase inhibitors with similar clinical effects will allow to develop aclinical protocol to avoid drug tolerance and provide a life-longtreatment.

Inhibitory agents of JNK, MLK or both, identified according to themethods of this invention, can be administered subcutaneously,intravenously, parenterally, intraperitoneally, intradermally,intramuscularly, topically, enteral (for example, orally), rectally,nasally, buccally, vaginally, by inhalation spray, by drug pump or viaan implanted reservoir in dosage formulations containing conventionalnon-toxic, physiologically (or pharmaceutically) acceptable carriers orvehicles.

In a specific embodiment, it may be desirable to administer the agentsof the invention locally to a localized area in need of treatment; thismay be achieved by, for example, and not by way of limitation, localinfusion during surgery, topical application, transdermal patches, byinjection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes orfibers.

In a specific embodiment when it is desirable to direct the drug to thecentral nervous system, techniques which can opportunistically open theblood brain barrier for a time adequate to deliver the drug therethrough can be used. For example, a composition of 5% mannitose andwater can be used. The present invention also provides pharmaceuticalcompositions. Such compositions comprise a therapeutically (orprophylactically) effective amount of the agent, and a physiologicallyacceptable carrier or excipient. Such a carrier includes, but is notlimited to, saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The carrier and composition can be sterile.The formulation should suit the mode of administration.

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions (for example, NaCl), alcohols, gumarabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin,carbohydrates such as lactose, amylose or starch, magnesium stearate,talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters,hydroxymethylcellulose, polyvinyl pyrolidone, etc. The pharmaceuticalpreparations can be sterilized and if desired, mixed with auxiliaryagents, for example, lubricants, preservatives, stabilizers, wettingagents, emulsifiers, salts for influencing osmotic pressure, buffers,coloring, flavoring and/or aromatic substances and the like which do notdeleteriously react with the active compounds.

The compositions, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,polyvinyl pyrollidone, sodium saccharine, cellulose, magnesiumcarbonate, etc.

The compositions can be formulated in accordance with the routineprocedure as a pharmaceutical composition adapted for intravenousadministration to human beings. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where the compositionis to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water, saline ordextrose/water. Where the composition is administered by injection, anampoule of sterile water for injection or saline can be provided so thatthe ingredients may be mixed prior to administration.

For topical application, there are employed as nonsprayable forms,viscous to semi-solid or solid forms comprising a carrier compatiblewith topical application and having a dynamic viscosity preferablygreater than water. Suitable formulations include but are not limited tosolutions, suspensions, emulsions, creams, ointments, powders, enemas,lotions, sols, liniments, salves, aerosols, etc., which are, if desired,sterilized or mixed with auxiliary agents, for example, preservatives,stabilizers, wetting agents, buffers or salts for influencing osmoticpressure, etc. The drug may be incorporated into a cosmetic formulation.For topical application, also suitable are sprayable aerosolpreparations wherein the active ingredient, preferably in combinationwith a solid or liquid inert carrier material, is packaged in a squeezebottle or in admixture with a pressurized volatile, normally gaseouspropellant, e.g., pressurized air.

The amount of agents which will be effective in the treatment of aparticular disorder or condition will depend on the nature of thedisorder or condition, and can be determined by standard clinicaltechniques. In addition, in vitro or in vivo assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances.

The invention will now be further and specifically described by thefollowing examples which are not intended to be limiting in any way. Allpublications cited herein are incorporated by reference in theirentirety.

EXAMPLES Example 1

Developing a Model for Neurodegeneration Based on Huntingtin Expression

To develop a cell-based system in which apoptosis can be induced,polyglutamine-expanded huntingtin was introduced into cell lines.

To assess whether polyglutamine-expanded huntingtin causes toxicity,full-length huntingtin expression constructs containing 16, 48 or 89 CAGrepeats were generated by assembly of a combination of RT-PCR productsfrom normal and human HD lymphoblast and plasmid cDNA clones IT16L andIT15B, which are described in HD Collaborative Research Group, Cell72:971-983 (1993).

To construct the huntingtin expression vector with 16, 48, or 89 CAGrepeats, the first third of the full-length construct was derived byligation of IT16LL (bp 932-3018) with three different PCR products (bp2401-3270, bp 637-1429 and 187-858). A 3027 bp cDNA fragment was removedfrom the resulting construct and ligated to corresponding sites in thecDNA clones IT15B (bp3024-10366). The CAG repeat size in the full lengthhuntingtin construct pFL16HD was 16. PCR products were generated fromthe genomic DNA of an adult patient with 48 CAG repeats and a juvenileonset case with 89 CAG repeats. These PCR products replaced thecorresponding region in pFL16HD to generate the pFL48HD and pFL89HD with48 and 89 CAG repeats, respectively. Colony hybridization and PCR wereused to identify the 48 and 89 CAG huntingtin clones and the positiveclones were verified by DNA sequence analysis.

The resulting constructs pFL16HD, pFL48HD or pFL89HD were transientlytransfected into 293 embryonic kidney cells, and expression ofhuntingtin was analyzed by immunoblotting using the anti-huntingtinmonoclonal antibody 4C8. Trottier, Y., et al., Nature Genet., 10:104-110(1995).

For transient expression of normal and polyglutamine-expandedhuntingtin, 50% to 60% confluent HN33 or 293 cells were washed once withserum free medium prior to transfection. Transfection was performed byusing lipofectin (Boehringer Mannheim) according to manufacturerinstructions and fetal bovine serum was added to the medium 12 hoursafter transfection to a final concentration of 1%. Sixty μg of plasmidwith 10 μl of lipofectin/60 mm plate was used in all transfectionexperiments. After 72 hours, the transfection medium was removed andreplaced by fresh medium with 1% fetal bovine serum. Forimmunoprecipitation and Western blotting, 293 cells were harvested 48-72hours after transfection and lysed in 1% NP-40 lysate buffer. For theimmunoprecipitation experiment, cell lysates were incubated with anaffinity-purified anti-N-terminus huntingtin polyclonal antibody 437 for4-6 hours. Liu, Y. F., et al., J. Biol. Chem., 272:8121-8124 (1997).Cell lysates or precipitated proteins were resolved on SDS-PAGE,transferred and immunoblotted with an anti-huntingtin monoclonalantibody 4C8. All three huntingtin constructs constitutively expressedthe huntingtin protein. Transfection of pcDNA1 (vector) or normalhuntingtin with 16 CAG repeats (pFL16HD) did not lead to toxicity inHN33 cells. In addition, DNA fragmentation was not detectable usingTdt-mediated dUTP-biotin nick end labeling (TUNEL) assay. However, cellproliferation was slightly suppressed.

The TUNEL assay was performed by using a TUNEL assay kit (BoehringerMannheim). HN33 cells were plated on a slide culture chamber. Transienttransfection as conducted as described above. Transfection medium wasremoved at specified times post-transfection. The cells were washed oncewith serum free medium, fixed with 4% paraformaldehyde and thenpermeabilized with 0.1% of Triton X-100. The TUNEL assay was performedas described in the manufacturer's instructions provided with the kit.

Expression of mutated huntingtin with 48 or 89 CAG repeats (pFL48HD orpFL89HD) induced cell toxicity in HN33 cells. Apoptosis was initiallyobserved between 20 and 24 hours after transfection by TUNEL staining.At 48 hours, about 75% of transfected cells were apoptotic.

A time-course of HN33 cell survival after transfection with pcDNA1,pFL16HD, pFL48HD or pFL89HD was performed forty eight hours aftertransfection, HN33 cells were fixed and stained with TUNEL. Living andapoptotic cells were counted in the high power field. The number ofliving cells in pcDNA1 transfectants is designated as 100%. FIG. 1indicates that, cell toxicity induced by transfection of pFL89HD wasslightly more severe than that mediated by pFL48HD, while transfectionof pcDNA1 or pFL16HD did not produce measurable neuronal toxicity. Eachdata point in FIG. 1 is an average of four independent experiments.

To further confirm that the polyglutamine-expanded huntingtin-inducedcell death described above was apoptotic, the interleukin 1β convertingenzyme (ICE) inhibitor zVAD-frm (Sarin et al., J. Exp. Med.184:2445-2449 (1996)) or CPP32 inhibitor zDEVD-frm (Rodgriguez et al.,J. Exp. Med. 184:2067-2072 (1996)) was added to the medium duringtransfection to a inhibitor concentration of 10 μg/mL. Both inhibitorswere obtained from Enzyme System Products, Inc. It was shown that ICEand CPP32 participated in apoptosis and that inhibitors zVAD-frm andzDEVD-frm blocked mutated huntingtin-mediated apoptotic cell death at 48hours post-transfection (FIG. 2). ICE cleaves inactive CPP32participated in apoptosis and that inhibitors zVAD-frm and zDEVD-frmblocked mutated huntington-mediated apoptotic cell death at 48 hourspost-transfection (FIG. 2). (Vaux et al., Proc. Natl. Acad. Sci. USA,93:2239-2244 (1996)). It was known that ICE cleaves inactive CPP32precursor thereby activating the enzyme. This result therefore suggeststhat expression of polyglutamine-expanded hungtingtin stimulates ICE,which in turn activates CPP32 to induce apoptotic cell death.

Example 2

Role of JNK in Neuronal Apoptosis

Whether expression of polyglutamine-expanded huntingtin inducesactivation of JNK was investigated. GST c-Jun (1-89 aa) was utilized asa substrate to measure JNK activity in cell lysates from HN33 cellstransfected with pcDNA1 (control), pFL16HD, pFL48HD or pFL89HD.

HN33 cells were lysed with 1% Triton buffer 16 hours after transfection.Cell lysates were incubated with glutatione-S-transferase (GST)-c-jun(1-89) fusion protein immobilized on glutatione sepharose beads toisolate JNK. These beads were resuspended in 30 μL kinase buffer. Thekinase reaction was performed at 30° C. for 30 minutes and then stoppedby adding SDS sample buffer to the reaction. The reaction was analyzedby Western blotting using a phospho (ser63)-specific c-jun antibody (NewEngland BioLabs).

A low level of JNK activation was observed in control cells.Transfection of a plasmid encoding normal huntingtin with 16 CAG repeatsdid not further increase the amount of serine phosphorylated GST c-Junand thus did not stimulate JNK activity. Expression of mutatedhuntingtin with 48 or 89 CAG repeats, however, significantly increasedthe levels of JNK activity. Serine phosphorylated GST c-Jun wasincreased 7- to 8-fold, similar to the level induced by 30 minutes ofultraviolet light irradiation. These results indicated thatpolyglutamine repeat expansion in huntingtin activated JNK in HN33 cells(FIG. 3).

Whether activation of JNK is responsible for polyglutamine-expandedhuntingtin-induced apoptotic cell death in HN33 cells was next examined.It was known that JNK is specifically activated by SEK1, which is adual-specificity kinase that phosphorylates both tyrosine and threonineresidues of JNK, thereby activiting it Sanchez et al., Nature, 380:75-79(1994)). A dominant negative mutant of SEK1 (K54R), known tospecifically block JNK activation (Lin et al., Science, 268:286-290(1995); Yan et al., Nature, 372:798-800 (1994)) was used.

HN33 cells were transiently transfected with various expressionplasmids, including the SEK1 and SEK1 (K54R) expression vectorsdescribed in Lin, A., et al., Science, 268:286-290 (1995). Forty-eighthours after transfection, the cells were fixed and subjected to theTUNEL assay as described above. The number of living cells transfectedwith pcDNA1 was designated at 100%. The data is summarized in FIG. 4.Each data point represents an average of three independent experiments.

Transient expression of wild-type or dominant negative SEK1 alone hadlittle effect on the proliferation and survival of HN33 cells.Co-expression of pcDNA1 with pEBG (SEK1 expression vector backbonecontrol plasmid) also did not lead to cell toxicity. However,co-transfection of wild-type SEK1 vector with pFL48HD or pFL89HD did notaffect neuronal toxicity induced by mutated huntingtin. Co-expression ofdominant negative mutant SEK1 (K54R) significantly prevented apoptoticcell death induced by mutated huntingtin. At 48 hours aftertransfection, about 25% to 30% of cells had undergone apoptosis,compared to about 75% of cells containing pFL48HD or pFL89HD alone.

The foregoing data showed that a specific inhibitor of JNK, SEK1 (K54R),can inhibit polyglutamine-expanded huntingtin-induced apoptosis and maybe a useful drug for treating a neurological condition. Thus it waspossible to test a compound's potential as a drug by contacting thecompound with a JNK inside a cell.

To determine whether the SEK1-JNK signal transduction pathway isinvolved in neuronal cell death induced by excitotoxicity induced byglutamate or kainate receptors, HN33 cells were transfected with pEBG(SEK1 expression vector backbone control plasmid), wild-type SEK1expression vector, or dominant negative mutant SEK1 (K54R) expressionvector. Forty-eight hours after transfection, the media covering thecells was replaced with fresh serum-free media supplemented without(control) or with 250 μM glutamate or 200 μM kainic acid (kainate).Cells were incubated in 37° C. for six hours. Then cells were fixed andstained with TUNEL. TUNEL negative cells (living cells) were counted.The results in FIG. 5 show that SEK1 (K54R) blocks apoptosis induced byglutamate or kainate receptor activation. Each data point represents theaverage of three independent experiments.

To determine further the role of JNK in glutamate-induced neuronalapoptosis, HN33 cells were treated with glutamate (250 μM) or kainicacid (200 μM) at room temperature for 1.5 minutes and then lysed in 1%Triton X-100 buffer. Cell lysates were incubated with GST-c-Jun (1-89)fusion protein immobilized on glutathione sepharose beads to isolateJNK. These beads were resuspended in 30 μl kinase buffer and the kinasereaction was performed at 30° C. for 30 minutes and 10 μl SDS samplebuffer was added to stop the reaction. The samples were resolved byelectrophoresis and transferred to a PVDF membrane (Millipore) and theJNK activity was analyzed by Western blotting using a phospho(Ser63)-specific c-Jun antibody (New England Biolabs). The results inFIG. 6 show that glutamate or kainate receptor activation inducedelevation of the JNK activity. Each data point represents an average ofthree independent experiments.

To determine the role of activation of the SEK1-JNK signal transductionpathway in Alzheimer's diseases, a cDNA fragment encoding thefull-length (Kang, J., et al., Nature, 325:733-736 (1987)) or the lastC-terminal 100 amino acid of amyloid precursor protein (APP) wasinserted into pcDNA1 and the resulting plasmid was designated asAPP-C-100. HN33 cells (60% of confluence) was transfected with pcDNA1(control), wild-type APP, APP-C-100 by using lipofectin (BoehringerMannheim) according to manufacture instruction. Thirty μl oflipofectin/50 mm plate was used in all transfection experiments. Twelveto seventy-two hours after transfection, HN33 cells were fixed with 4%paraformaldehyde and permeabilized with 0.1% of Triton X-100. The TUNELstaining was performed by using a TUNEL staining kit (BoehringerMannheim) as described in manufacture instructions provided with thekit. TUNEL negative cells were counted under light microscope. Theresults in FIG. 7 show that expression of APP-C-100 induced rapidneuronal apoptosis. Cell death was initially observed between twenty totwenty-four hours after transfection and at seventy-two hours, all cellswere apoptotic, while expression of pcDNA1 (control) or wild-type APPdid not induce neuronal cell death.

To determine the role of the JNK activity in neuronal cell death inducedby APPC-100, HN33 cells were transiently transfected with pcDNA1,wild-type APP or APPC-100 using lipofectin as described above. Aftereighteen hours, cells were lysed in 1% Triton C-100 buffer. Cell lysateswere incubated with GST-c-Jun (1-89) fusion protein immobilized onglutathione sepharose beads to isolate JNK. These beads were resuspendedin 30 μl kinase buffer and the kinase reaction was performed at 30° C.for 30 minutes and 10 μl SDS sample buffer was added to stop thereaction. The JNK activity was analyzed by Western blotting using aphospho (Ser63)-specific c-Jun antibody (New England BioLabs). Theresults in FIG. 8 show that expression of APP-C-100 but not wild-typeAPP stimulated the JNK activity in HN33 cells.

To further determine the role of activation of the JNK activation inAlzheimer's diseases, HN33 cells were transfected with pcDNA1, wild-typeAPP expression vector, or mutant SEK1 expression vector using lipofectin(Boehringer Mannheim) according to manufacture instruction. Forty-eighthours after transfection, cells were fixed and stained with TUNEL. TUNELnegative cells (living cells) were counted. The results in FIG. 9 showthat SEK1 (K54R) blocks apoptosis induced by expression of APP-C-100.

Taken together, these results indicate the elevation of the JNK activityis a common cause of neuronal cell death, regardless of the cause. Sinceexcitotoxicity is a final common pathway for neuronal loss inneurodegenerative disease as well as in acute insults, inhibition of theJNK activity will prevent neuronal death in these neurologicalconditions.

Example 3

Role of MLK in Neuronal Apoptosis

A kinase dead version of MLK2 was generated by introduction of A-G pointmutation at position 651 (codon AGG to GAG) by overlapping extensionusing polymerase chain reaction with mutated oligonucleotides, to resultin amino acid substitution of K to E in the ATP binding loop of the MLK2kinase domain. Such a point mutation leads to total loss of kinaseactivity of MLK2 and a kinase dead version of MLK2 will act as adominant mutant and inhibit MLK2 activation-mediated actions. Tibbles,L. A., et al., EMBO. J., 15:7026-7036 (1996). The cDNA fragment ofwild-type or kinase dead MLK2 was inserted into pRK5CMV with aC-terminal myc tag. Nagata, K., et al., EMBO. J., 17:149-158 (1998).

To examine whether expression of MLK2 induces neuronal cell death, HN33cells were transfected with pRK5CMV, wild-type or kinase dead MLK2expression vector using lipofectin (Boehringer Mannheim) according tomanufacture instruction. Forty-eight hours after transfection, cellswere fixed and stained with TUNEL, as described above. TUNEL negativecells (living cells) were counted. The results in FIG. 10 show thatexpression of MLK2 induced apoptosis in HN33 cells while expression ofthe kinase dead version of MLK2 did not generate any cell toxicity.

To determine whether the role of the SEK1-JNK signal transductionpathway in neuronal cell death induced by expression of MLK2, HN33 cellswere co-transfected with pEBG+pRK5CMV (control), wild-type SEK1expression vector+wild-type MLK2 expression vector, or dominant negativemutant SEK1 (K54R) expression vector+wild-type MLK2 expression vector.Forty-eight hours after transfection, cells were fixed and stained withTUNEL. TUNEL negative cells (living cells) were counted. The results inFIG. 11 show that SEK1 (K54R) attenuated apoptosis induced by MLKexpression.

To determine the role of MLK2 in neuronal loss in Huntington's disease,HN33 cells were co-tranfected with pEBG+pRK5CMV (control), normalhuntingtin expression vector with 48 or 89 CAG repeats+kinase dead MLK2expression vector. Forty-eight hours after transfection, cells werefixed and stained with TUNEL. TUNEL negative cells (living cells) werecounted. The results in FIG. 12 show that co-expression of kinase deadMLK2 blocked neuronal apoptosis induced mutated huntingtin anddemonstrated that the MLK-associated activity was involved in neuronalloss in Huntington's diseases.

The potential association of huntingtin with MLK2 was examined in 293cells transfected with MLK2, normal huntingtin, orpolyglutamine-expanded huntingtin in 293 cells at 48 hours aftertransfection. Expression of normal or mutated huntingtin failed toactivate JNKs. Since 293 cells are rich in huntingtin (Liu, Y. F., etal., J Biol. Chem., 272:8121-8124 (1997)), the interaction of MLK2 withnormal huntingtin in 293 cells was examined. C-Myc tagged MLK2 wastransiently expressed in 293 cells, and MLK2 was precipitated withanti-c-myc tag 9E10 antibody (Santa Crutz).

C-myc-tagged MLK2 was transiently expressed in 293 cells, as describedabove, 48-72 hours after transfection, 293 cells were harvested andlysed in 1% NP-40 lysate buffer. Cell lysates were incubated with ananti-N-terminus huntingtin antibody 437 or 9E10 for 4-6 hours. Theprecipitated proteins were resolved on SDS-PAGE, transferred, andimmunoblotted with an anti-huntingtin monoclonal antibody 4C8, oranti-c-myc antibody 9E10.

In cell lysates from 293 cells transfected without or with pRK5CMVcontrol vector described above, 9E10 failed to co-precipitatehuntingtin. However, when the 293 cells were transfected with c-myctagged MLK2, huntingtin was easily detected in both 9E10 and 4C8(anti-huntingtin) immunoprecipitates. Thus, MLK2 is associated withhuntingtin.

Conversely, whether an anti-huntingtin antibody precipiated MLK2 wasdetermined. Cell lysates from 293 cells with or without transfection ofa MLK2 expression vector were incubated with 437, an anti-huntingtinantibody, or 9E10. MLK2 was detectable in both 9E10 and 437immunoprecipitates of 293 cell lysates transfected with MLK2. Innon-tranfected 293 cells, MLK2 was not found in either 9E10 or 437immunoprecipitates. These results demonstrate that huntingtin isassociated with MLK2 in vivo.

Next, we tried to examine the novel association of MLK2 with mutatedhuntingtin containing 48 CAG repeats in 293 cells. Expression of MLK2 ormutated huntingtin alone did not generate any cell toxic effect.Co-expression of MLK2 with normal huntingtin containing 16 CAG repeatsalso did not produce any cell toxicity while co-expression of mutatedhuntingtin with induced rapid cell death.

To determine whether the role of MLK2 in neuronal cell death induced byglutamate or kainate receptor activation, HN33 cells were transfectedwith pRK5CMV (control) or kinase dead MLK2 expression vector.Forty-eight hours after transfection, the media covering the cells wasreplaced with fresh serum-free media supplemented without (control) orwith 250 μM glutamate or 200 μM kainate. Cells were incubated in 37° C.for six hours, fixed, and then stained with TUNEL. TUNEL negative cells(living cells) were counted. The results in FIG. 13 show that kinasedead MLK2 blocks apoptosis induced by glutamate or kainate receptoractivation in HN33 cells.

To determine further the role of activation of the MLK activation inAlzheimer's diseases, HN33 cells were co-transfected with pcDNA1,wild-type expression vector, with kinase dead MLK2 expression vector.Forty-eight hours after transfection, cells were fixed and stained withTUNEL. TUNEL negative cells (living cells) were counted. The results inFIG. 14 show that kinase dead MLK2 blocks apoptosis induced byexpression of APP-C-100.

Taken together, these results shown that the MLK-associated activitymediated neuronal degeneration in Huntington's disease, Alzheimer'sdisease and excitotoxicity induced by glutamate or kainate receptoractivation. Since excitotoxicity is a final common pathway for neuronalloss in neurodegenerative disease as well as in acute insults,inhibition of the MLK2 activity will prevent neuronal death in theseneurological conditions.

Example 4

Preparation and Purification of GST Fusion Proteins

Glutathione-S-transferase (GST)) c-Jun fusion protein was utilized asthe substrate for JNK3 kinase assay and GST SEK1 fusion protein wasutilized as the substrate for MLK2 kinase assay. To generate GST-c-Junor GST-SEK (K54R) fusion proteins, the cDNA fragment encoding theN-terminus of c-Jun 1-89 amino acid residues, or the full-length cDNA ofSEK1, was subcloned into a pGEX vector. The vector was subsequentlytransformed into E. coli and the E. coli carrying pGEX-Jun or PGX-SEK1was grown in LB medium. Expression of these GST fusion proteins wasinduced by 0.1 mM isopropyl-b-D-thiogalactopyraside (IPTG). Cells werepelleted and resuspended in 1/60 culture volume of MT PBS (150 mM NaCl,16 mM Na₂HPO₄, 4 mM NaH₂PO₄, pH 7.3) and then lysed by mild sonicationafter adding Triton X-100 to the final concentration of 1% followed bycentrifugation at 10,000×g for 5 min at 4° C. The supernatant was mixedat room temperature in a 50 ml polypropylene tube on a rotating platformwith 1-2 ml 50% glutathione agarose beads. After absorption for 2 min,beads were collected by brief centrifugation at 500×g and washed threetimes with 50 ml MT PBS. The c-Jun or SEK1 GST fusion proteins wereeluted by competition with free glutathione using 2×2 min washes twicewith the same buffer, and stored. The purified GST fusion proteins werestored at −80° C. in MT PBS at 4° C. as a 50% solution.

Example 5

Transfection and JNK in vitro Kinase Assay

For an in vitro JNK kinase assay, including JNK1, JNK2 and JNK3,isolated JNK protein was used. Isolation may be accomplished bychromatographic purification from tissue or molecular transfection ofhost cells followed by isolation. For molecular isolation of JNK, a fulllength cDNA of either JNK1, JNK2 or JNK3 glutathione-S-transferase (GST)fusion construct was inserted into pGEX-2T vector and expressed in ahost, such as a bacterial cell (e.g., DH1 cell). The fusion protein wasthen purified from the host cell using standard techniques known tothose skilled in the art. Isolation of the GST-fusion protein wasaccomplished using a glutathione affinity column. Approximately 5 μL ofglutathione-Sepharose was used to recover the fusion protein. The resinwas washed three times with lysis buffer (20 mM Tris-HCl, pH 8.0, 2 mMEDTA, 50 mM P-glycerophosphate, 1 mM Na₂PO₄, 1% Triton X-100, 10%glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/mL leupeptinand 10 μg/mL aproptinin) and twice with detergent-free lysis buffer. TheGST-fusion protein was eluted from the resin with 100 μL of 5 mMglutathione in a detergent-free lysis buffer.

The in vitro kinase reaction was carried out at 30° C. for 15 minutes ina kinase mixture containing 0.5 μg of GST-JNK3, 1 μg of GST-c-Jun, 2.5mM ATP and 5 μCi of [γ-³²P] ATP with or without a test compound in 20 μLof kinase buffer (20 mM HEPES, pH 7.5, 15 mM MgCl₂, 15 mMβ-glycerophosphate, 0.1 mM Na2PO₄, 2 mM dithiothreitol). The reactionwas terminated by adding 20 μL of SDS sample buffer (Laemmeli buffer).The phosphorylation was detected by SDS-PAGE, and the amount of ³²Pincorporated was quantified with an image analyzer. Effectiveness of thetest compound as an inhibitor of JNK correlated with inhibitor of ³²Pincorporated GST-c-Jun.

Example 6

Transfection and MLK2 or MLK3 in vitro Kinase Assay

For an in vitro MLK2 or 3 kinase assay, isolated MLK2 protein is used.Isolation may be accomplished by chromatographic purification fromtissue or molecular transfection of host cells followed by isolation.For molecular isolation of MLK, a full length cDNA of either MLK2 orMLK3 glutathione-S-transferase (hereinafter GST) fusion construct wasinserted into pEGB vector and expressed in 293 host cells usinglipofectin, as described above for transfection of mutated huntingtininto HN33 cells. Forty eight hours after transfection, the cells werelysed using 0.5 ml at a lysis buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA,50 mM β-glycerophosphate, 1 mM Na₂VO₄, 1% Triton X-100, 10% glycerol, 1mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml leupeptin and 10 μg/mlaproptinin). The fusion protein was then purified from the host cellusing standard techniques known to those skilled in the art. Isolationof the GST-fusion protein was accomplished using a glutathione affinitycolumn. Approximately 5 μL of glutathione-Sepharose was used to recoverthe fusion protein. The resin was washed three times with lysis bufferand twice with detergent-free lysis buffer. The GST-fusion protein waseluted from the resin with 100 μL of 5 mM glutathione in adetergent-free lysis buffer.

The in vitro kinase reaction was carried out at 30° C. for 45 minutes ina 20 μL kinase mixture containing 1 μg of GST-MLK2 or MLK3, 1 μg ofGST-SEK1 (K54R), 2 mM ATP and 5 μCi or [γ-³²P] ATP in the absence orpresence of a test compound in the kinase buffer (20 mM HEPES, pH 7.5,15 mM MgCl₂, 15 mM β-glycerophoshate, 0.1 mM Na₂VO₄, 2 mMdithiothreitol). The reaction was terminated by adding 20 μL of SDSsample buffer (Laemmeli buffer). The phosphorylation was detected bySDS-PAGE, and the amount of ³²P incorporated was quantified with animage analyzer. Effectiveness of the test compound as an inhibitor ofMLK2 or MLK3 correlated with inhibition of ³²P incorporated GST-SEK1(K54R).

Example 7

MLK2 Immunocomplex Kinase Assay

An MLK2 immunocomplex kinase assay was used to screen for the MLK 2 or 3inhibitor and to determine the specificity of the identified MLKinhibitor. The full-length cDNA of MLK 2 or 3 was inserted into pRK5vector and tagged with c-myc at the C-terminus. 293 embryonic cells weregrown in DMEM with 10% FBS in a 6-well dish and then were transfectedwith pRK5MLK2 using lipofectin as described above. Forty-eight hoursafter transfection, cells were lysed with 0.5 ml of a lysis buffer (20mM Tris-HC1, pH 8.0, 2 mM EDTA, 50 mM β-glycerophosphate, 1 mM Na₂VO₄,1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride(PMSF), 10 μg/ml leupeptin, and 10 μg/ml aproptinin) and the lysateswere incubated with anti-c-myc tag antibody 9E10 for 1 hour and 20 μl ofa mixture (50×50) or protein G and A-Sepharose resin was add andcontinued to incubate for another 2-4 hours. The resin was washed twicewith lysis buffer and twice with detergent-free lysis buffer. The pelletwas then resuspended in 20 μl of kinase buffer (20 mM HEPES, pH 7.5, 15mM MgCl₂, 15 mM β-glycerophosphate, 0.1 mM Na₂VO₄, 2 mM dithiothreitol,25 μM ATP, 5 μCi of [γ⁻³²P]ATP, and 0.5 μg of SEK1 (K54R) GST fusionproteins in the presence or absence (control) of 1 nM to 10 μM of amixture of compounds or the identified compounds. The kinase reactionwas carried out at 30° C. for 20 min. and stopped by adding 20 μl of SDSsample buffer. The phosphorylation is detected by SDS-PAGE, and theamount of ³²P incorporated is quantified with an image analyzer. If acompound is effective to inhibit MLKs, the amount of ³²P incorporated issignificantly reduced or totally inhibited.

Example 8

96-Well Cell Based Assay

HN33 cells (˜60% of confluence), plated on a 96-well plate, were grownin DMEM-F12 medium supplemented with 10% of fetal bovine serum (FBS).Prior to transfection, the medium was removed and cells were washed withserum-free medium once and 50 μl of DMEM-F12 medium with 1% of FBS wasadded. The full-length huntingtin expression plasmid containing 16(control), 48 or 89 CAG repeats or lipofectin solution were diluted withHBS first and mixed 1×1 volume and the mixture was incubated at roomtemperature for 15 min. Ten μl of the DNA-lipofectin mixture was addedto the culture medium. A mixture of compound from a chemical library wasadded 6 hours after transfection. Twelve hours after transfection,additional FBS was added to the final concentration of 10%. Forty-eighthours after transfection, cells were washed with PBS once and fixed with4% paraformaldehyde dissolved in PBS and permeabilized with 1% TritonX-100 in PBS containing 1% sodium citrate. Cell were then rinsed twicewith PBS containing 1% BSA and cells were incubated with 25 μl of TUNELreaction solution for 1 hour at a 37° C. cell culture incubator. Cellwere rinsed with PBS for three times and 25 μl of converter-AP solutionwas added and cells are returned to the incubator and incubated for 30in. Cells were rinsed three times with PBS with 1% BSA and cells wereincubated in a BCIP solution for 1-5 min at room temperature and rinsedthree times with PBS and analyzed under light microscope.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1. A method for assessing a compound's ability to inhibit JNK activityand prevent neuronal cell death comprising: a) incubating the compoundin the presence of isolated JNK and a JNK substrate, under conditionssufficient for JNK kinase activity; and b) determining the presence oramount of phosphorylated JNK substrate; wherein a change in the presenceor amount of phosphorylated JNK substrate, where compared to incubatingisolated JNK with the JNK substrate absent the compound, is indicativeof the compound's ability to inhibit the JNK kinase activity; c)contacting the compound having an ability to inhibit JNK activity withneuronal cells transfected with a mutated protein or treated with aneurotoxin to induce apoptosis, wherein the mutated protein comprisespolyglutamine stretch-expanded huntingtin or C-terminal 100 amino acidsof amyloid precursor protein; and d) comparing the occurrence ofapoptosis in the neuronal cells in the presence of the compound with theoccurrence of apoptosis in the neuronal cells in the absence of thecompound, wherein the compound having an ability to inhibit the JNKactivity has the ability to prevent neuronal cell death when theoccurrence of apoptosis in the neuronal cells in the presence of thecompound is less than the occurrence of apoptosis in the neuronal cellsin the absence of the compound wherein JNK is JNK1, JNK2, orcombinations thereof.
 2. The method of claim 1, wherein the JNKsubstrate includes c-Jun and a phosphate donor.
 3. The method of claim1, wherein the phosphorylated JNK substrate of step (b) isphosphorylated c-Jun.
 4. A method for assessing a compound's ability toinhibit JNK activity and prevent neuronal cell death comprising: a)incubating the compound in the presence of isolated JNK and a JNKsubstrate, under conditions sufficient for JNK kinase activity; and b)determining the presence or amount of phosphorylated JNK substrate;wherein a change in the presence or amount of phosphorylated JNKsubstrate, where compared to incubating isolated JNK with the JNKsubstrate absent the compound, is indicative of the compound's abilityto inhibit the JNK kinase activity; c) contacting the compound having anability to inhibit JNK activity with neuronal cells, said neuronal cellsobtained from neuronal cell line HN33; and d) comparing the occurrenceof apoptosis in the neuronal cells in the presence of the compound withthe occurrence of apoptosis in the neuronal cells in the absence of thecompound, wherein the compound having an ability to inhibit the JNKactivity has the ability to prevent neuronal cell death when theoccurrence of apoptosis in the neuronal cells in the presence of thecompound is less than the occurrence of apoptosis in the neuronal cellsin the absence of the compound wherein JNK is JNK1, JNK2, orcombinations thereof.